Figure 3.

Group I ribozyme reactions used in this work. (A) L-21 ScaI cleavage reaction (31) used for kinetics and binding studies. In the presence of guanosine, the bound substrate oligonucleotide is cleaved by the ribozyme into two fragments in a reaction that can be monitored kinetically (22). Product binding affinity can be studied by gel shift analysis (21). (B) L-21 G414 ligation reaction used for NAIM and NAIS experiments, in which G414, bound in the G-binding site, attacks a 3′-labeled substrate oligonucleotide. The reaction is analogous to the reverse of the second step of splicing (23), in which the AAAAAA* is covalently transferred onto the 3′ ends of the active ribozyme variants.